We continued our work on the membrane biology of pathogenic processes. Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated with simultaneous recordings of whole-cell admittance and fluorescence, in addition to rapidly frozen, freeze-substitution, thin section electron microscopy. Upon triggering with acid solution conformational changes in HA expressed by CHO cells and bound to red blood cells, the previously separated membranes developed a multiplicity of 10-50 nm points of membrane coalescence which did not enlarge further or combine. Physiological measurements showed fast lipid dye mixing between cells after acidification, and small fusion pores developed in a significant fraction of the cell pairs. Those GPI-HA induced pores behaved qualitatively similar to HA induced pores but never expanded. Lipid mixing was detected either prior to or during pore opening. These findings indicate that the transmembrane domain of HA, though not essential for the pore formation, is strictly required to compose the fusion complex in a way to ensure the pore opening and its subsequent expansion. Second, the energetics underlying the expansion of fusion pores, which determines pore growth, was studied. For two homogeneous fusing membranes under different tensions, pore growth can be quantitatively described by treating the pore as a quasi-particle that moves in a medium with a viscosity determined by that of the membranes. This treatment explains how increases in tension through osmotic swelling of vesicles cause enlargement of pores between the vesicles and planar bilayer membranes. The calculations also show that for biological fusion, pore expansion can be regulated by pore length: the membrane mechanics of pore lengthening is an energetically favored process.Third, membrane permeability changes in apoptosis were studied. Release of proteins through the outer mitochondrial membrane is a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro, and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability, and decreased the stability of the membrane, regardless of the origin of the protein. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage and concentration-dependent manner. Bcl-xL did not protect against Bax induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e. the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, forming a pore -- the apoptotic pore -- large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole. - influenza, viral envelope, hemagglutinin, membrane tension, bax, bcl, mitochondria, phospholipid bilayer, fusion pore, hemifusion.